"Milk on Ice": A detailed analysis of Ernest Shackleton's century-old whole milk powder in comparison with modern counterparts.

Whole milk powder (WMP) manufactured in New Zealand in 1907 was sent to the Antarctic continent with the Shackleton-led British Antarctic Expedition from 1907 to 1909. This powder was stored at ambient conditions at Shackleton's Hut at Cape Royds, Antarctica, for over 100 yr before a sample was collected on behalf of Fonterra by the Antarctic Heritage Trust. Having spent most of its existence both dried and in frozen storage, any deleterious reactions within the WMP would have been markedly retarded. The composition and some properties of the roller-dried Shackleton's WMP are reported along with those of 2 modern spray-dried New Zealand WMP. The Shackleton powder was less white and more yellow than the modern WMP and was composed of flakes rather than agglomerated particles, consistent with that expected of a roller-dried powder. Headspace analysis showed lipolytic and oxidative volatile compounds were present in the Shackleton WMP, indicting some deterioration of the milk either before powder manufacture or on storage of the finished product. On a moisture-free basis, the Shackleton WMP had higher protein, higher fat (with a markedly higher free fat level), higher ash, and a lower lactose level than the modern WMP. The lysine level was lower in the Shackleton WMP compared with the spray-dried powders, whereas the fatty acid composition was relatively similar. The sodium level was markedly higher in the Shackleton WMP compared with the spray-dried powder, which is probably due to the addition of an alkaline sodium salt to adjust the pH of the milk before roller drying. Lead, iron, and tin levels were markedly higher in the Shackleton WMP compared with the spray-dried powders, possibly due to the equipment used in powder manufacture and the tin-plated cases used for storage. The proteins in the Shackleton WMP were more lactosylated than in the spray-dried powders. The Shackleton WMP had a higher ratio of κ-casein A to B variants and a higher ratio of ß-lactoglobulin B to A variants than the spray-dried powders, whereas the αS1-casein, ß-casein, αS2-casein, and α-lactalbumin protein variants were similar in all powders. The total phospholipid content was markedly lower in the Shackleton WMP than the spray-dried powders, primarily due to a lower phosphatidylethanolamine concentration. The molecular species distributions within the phospholipid classes were generally similar in the 3 powders. Claims are sometimes encountered that the milk of today is different from that consumed by previous generations. However, this comparative study has shown that the Shackleton WMP was generally similar to modern WMP. Although differences in some components and properties were observed, these were attributable to the manufacturing equipment and processes used in the pioneering years of WMP manufacture.

Hemp globulin forms colloidal nanocomplexes with sodium caseinate during pH-cycling.

Seed from industrial hemp (Cannabis sativa L.) contains around 25% protein (mainly globulins) which is easily digested, but the low solubility of hemp globulins (HG) limits their application in many food systems. In this study, the solubility of HG was improved by blending HG with sodium caseinate (SC) and treating with a pH-cycling process. The pH-cycling involved adjusting the pH to 12 and reacting for 1 hr, followed by neutralisation to pH 7. Nanoparticles composed of HG and SC (Z-average diameter ≈ 130 nm) were formed after the pH-cycling, and the solubility of HG increased to > 80% when there was more than 1% of SC for 1% of HG. These HG|SC nanoparticles were monodisperse (PDI < 0.17) and ζ-potential was ≈ -17 mV. Hydrogen bonding is the main forces that assembles HG|SC nanoparticles because the nanoparticles dissociated by heat treatment (up to 60 °C) or urea, which is an effective hydrogen bond breaker. HG|SC nanoparticles will aggregate irreversibly above 60 °C, possibly due to thiol-disulphide exchange. The nanoparticles were heat-stable as the Z-average diameter was only 229 nm after heating (90 °C, 30 min). N-ethylmaleimide blocked free thiol groups on HG and resulted in less disulphide-linked HG aggregation after pH- cycling, which in turn lead to smaller HG|SC nanoparticles and a bimodal particle size distribution, indicating the importance of disulphide bond for the formation of monodisperse HG|SC nanoparticles. The soluble and heat-stable HG|SC nanoparticles could be used to increase the hemp protein content in beverages and emulsions.

Concentrated Pickering emulsions stabilised by hemp globulin-caseinate nanoparticles: tuning the rheological properties by adjusting the hemp globulin : caseinate ratio.

Industrial hemp (Cannabis sativa L.) is an underutilised novel protein source. However, the utilisation of hemp seed protein is limited by its low solubility in water. Soluble nanoparticles were made by complexing hemp globulin (HG) with sodium caseinate (SC) via a pH-cycling method. Oil-in-water Pickering emulsions were made with these co-assembled protein nanoparticles. The emulsions were composed of 70% oil phase and 30% water phase (v/v), and contained 2% protein (w/v, pure SC or HG-SC nanoparticles with an HG : SC ratio of 1 : 2 or 1 : 1). All emulsions were stable during 21 days of storage, as there was no phase separation, coalescence or flocculation. At day 0, all emulsions were solid-like (G' > G'') regardless of the protein composition. The rheological properties of the emulsions during storage could be tuned by controlling the HG : SC ratio in the HG-SC nanoparticles, i.e. the emulsions became more solid-like over time when there was more HG in the nanoparticles. In contrast, emulsions stabilised by pure SC became more liquid-like during storage. The internal structure and interactions within the emulsions were evaluated by fitting frequency sweep test data according to a co-operative theory of flow. The result suggested that the solid-like emulsion resulted from stronger short-range interactions between flocs of oil droplets, which developed during storage when there was more HG in the HG-SC nanoparticles, and not from the formation of a three-dimensional network. These HG-SC nanoparticles can be used to control the rheological properties of an emulsion during its shelf life.

Age Gelation, Sedimentation, and Creaming in UHT Milk: A Review.

Demand for ultra-high-temperature (UHT) milk and milk protein-based beverages is growing. UHT milk is microbiologically stable. However, on storage, a number of chemical and physical changes occur and these can reduce the quality of the milk. These changes can be sufficiently undesirable so as to limit acceptance or shelf life of the milk. The most severe changes in UHT milk during storage are age gelation, with an irreversible three-dimensional protein network forming throughout, excessive sedimentation with a compact layer of protein-enriched material forming rapidly at the bottom of the pack, and creaming with excessive fat accumulating at the top. For age gelation, it is known that at least two mechanisms can lead to gelation during storage. One mechanism involves proteolytic degradation of the proteins through heat-stable indigenous or exogenous enzymes, destabilizing milk and ultimately forming a gel. The other mechanism is referred to as a physico-chemical mechanism. Several factors are known to affect the physico-chemical age gelation, such as milk/protein concentration, heat load during processing (direct compared with indirect UHT processes), and milk composition. Similar factors to age gelation are known to affect sedimentation. There are relatively few studies on the creaming of UHT milk during storage, suggesting that this defect is less common or less detrimental compared with gelation and sedimentation. This review focuses on the current state of knowledge of age gelation, sedimentation, and creaming of UHT milks during storage, providing a critical evaluation of the available literature and, based on this, mechanisms for age gelation and sedimentation are proposed.

Ultrasonication of reconstituted whole milk and its effect on acid gelation.

Ultrasonication (US) of whole milk at 22.5kHz and 50W homogenized fat globules. Extended US without temperature control (attaining >90°C at longest times), or with control at temperatures ⩾60°C caused denaturation of the whey proteins and aggregation of the fat globules and proteins. Acidification of US milk produced gels with increased firmness and reduced gelation times compared to untreated milk. Below 60°C, US of milk produced acid gels with very high firmness without whey protein denaturation; the firmness was similar to gels from heated whole milk. Extensive US without temperature control or with control at ⩾60°C decreased acid gel firmness compared to shorter times or lower temperatures. Higher acid gel firmness could be achieved by subjecting the milk to separate heat (80°C/30min) and US treatment (at 20°C) before acidification when compared with either heating or US alone. This was independent of the order of heating and US treatment.

Reassociation of dissociated caseins upon acidification of heated pH-adjusted skim milk.

Milk was heated at different pH (pH 6.5-7.1) and temperatures (20-120 °C/10 min). This resulted in different levels of casein and denatured whey proteins to be distributed between the colloidal and serum phases. The milks were subsequently acidified and the distribution of protein between colloidal and serum was monitored at different pH. On acidification to pH 5.4, the serum phase caseins and denatured whey proteins partially reassociated with the caseins, although a complex behaviour was observed at ∼ pH 5.4 where additional casein dissociation occurred in some samples. At pH below 5.4 the caseins and denatured whey proteins rapidly aggregated. No separate aggregation of κ-casein/denatured whey protein complexes or κ-casein depleted micelles was observed. The earlier gelation of milks heated at higher pH was likely to be due to the destabilisation of the entire milk protein system rather than a preferential aggregation of the serum phase proteins.

Complex coacervates of lactotransferrin and ß-lactoglobulin.

HYPOTHESIS: Oppositely charged proteins should interact and form complex coacervates or precipitates at the correct mixing ratios and under defined pH conditions. EXPERIMENTS: The cationic protein lactotransferrin (LF) was mixed with the anionic protein ß-lactoglobulin (B-Lg) at a range of pH and mixing ratios. Complexation was monitored through turbidity and zeta potential measurements. FINDINGS: Complexation between LF and B-Lg did occur and complex coacervates were formed. This behaviour for globular proteins is rare. The charge ratio's of LF:B-Lg varies with pH due to changing (de) protonation of the proteins. Nevertheless we found that the complexes have a constant stoichiometry LF:B-Lg=1:3 at all pH's, due to charge regularization. At the turbidity maximum the zeta potential of complexes is close to zero, indicating charge neutrality; this is required when the complexes form a new concentrated liquid phase, as this must be electrically neutral. Complexes were formed in pH region 5-7.3. On addition of salt (NaCl) complexation is diminished and disappears at a salt concentration of about 100 mMol. The coacervate phase has a very viscous consistency. If we consider the proteins as colloidal particles then the formed complex coacervate phase may have a structure that resembles a molten salt comparable to, for example, AlCl3.

Interactions of casein micelles with calcium phosphate particles.

Insoluble calcium phosphate particles, such as hydroxyapatite (HA), are often used in calcium-fortified milks as they are considered to be chemically unreactive. However, this study showed that there was an interaction between the casein micelles in milk and HA particles. The caseins in milk were shown to bind to the HA particles, with the relative proportions of bound ß-casein, αS-casein, and κ-casein different from the proportions of the individual caseins present in milk. Transmission electron microscopy showed no evidence of intact casein micelles on the surface of the HA particles, which suggested that the casein micelles dissociated either before or during binding. The HA particles behaved as ion chelators, with the ability to bind the ions contained in the milk serum phase. Consequently, the depletion of the serum minerals disrupted the milk mineral equilibrium, resulting in dissociation of the casein micelles in milk.

Glycation as a Tool To Probe the Mechanism of ß-Lactoglobulin Nanofibril Self-Assembly.

In this study we investigated the effects of different levels of glucosylation and lactosylation on ß-Lg self-assembly into nanofibrils at 80 °C and pH 2. Fibrils in heated samples were detected with the thioflavin T assay and transmission electron microscopy, while SDS-PAGE was used to investigate the composition of the heated solutions and fibrils. Glycation had different effects in the nucleation and growth phases. The effect of glycation on the nucleation phase depended on the degree of glycation but not the sugar type, whereas both the type of sugar and the degree of glycation affected the rate of fibril growth. Glycation by either sugar strongly inhibited self-assembly in the growth phase, and lactosylation produced a much stronger effect than glucosylation. We suggest that the varying glycation susceptibility of different lysine residues can explain these observations. The large, polar sugar residues on the glycated fibrillogenic peptides may inhibit fibril assembly by imposing steric restrictions and disrupting hydrophobic interactions.

Modulating ß-lactoglobulin nanofibril self-assembly at pH 2 using glycerol and sorbitol.

ß-Lactoglobulin (ß-lg) forms fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). When formed at protein concentrations <3%, these fibrils are made up of peptides produced from the acid hydrolysis of the ß-lg monomer. The present study investigated the effects of the polyhydroxy alcohols (polyols) glycerol and sorbitol (0-50% w/v) on ß-lg self-assembly at pH 2. Glycerol and sorbitol stabilize native protein structure and modulate protein functionality by preferential exclusion. In our study, both polyols decreased the rate of ß-lg self-assembly but had no effect on the morphology of fibrils. The mechanism of these effects was studied using circular dichroism spectroscopy and SDS-PAGE. Sorbitol inhibited self-assembly by stabilizing ß-lg against unfolding and hydrolysis, resulting in fewer fibrillogenic species, whereas glycerol inhibited nucleation without inhibiting hydrolysis. Both polyols increased the viscosity of the solutions, but viscosity appeared to have little effect on fibril assembly, and we believe that self-assembly was not diffusion-limited under these conditions. This is in agreement with previous reports for other proteins assembling under different conditions. The phenomenon of peptide self-assembly can be decoupled from protein hydrolysis using glycerol.

Protein composition of different sized casein micelles in milk after the binding of lactoferrin or lysozyme.

Casein micelles with bound lactoferrin or lysozyme were fractionated into sizes ranging in radius from ∼50 to 100 nm. The κ-casein content decreased markedly and the αS-casein/ß-casein content increased slightly as micelle size increased. For lactoferrin, higher levels were bound to smaller micelles. The lactoferrin/κ-casein ratio was constant for all micelle sizes, whereas the lactoferrin/αS-casein and lactoferrin/ß-casein ratio decreased with increasing micelle size. This indicates that the lactoferrin was binding to the surface of the casein micelles. For lysozyme, higher levels bound to larger casein micelles. The lysozyme/αS-casein and lysozyme/ß-casein ratios were nearly constant, whereas the lysozyme/κ-casein ratio increased with increasing micelle size, indicating that lysozyme bound to αS-casein and ß-casein in the micelle core. Lactoferrin is a large protein that cannot enter the casein protein mesh; therefore, it binds to the micelle surface. The smaller lysozyme can enter the protein mesh and therefore binds to the more charged αS-casein and ß-casein.

Coacervates of lactotransferrin and ß- or κ-casein: structure determined using SAXS.

Lactotransferrin (LF) is a large globular protein in milk with immune-regulatory and bactericidal properties. At pH 6.5, LF (M = 78 kDa) carries a net (calculated) charge of +21. ß-Casein (BCN) and κ-casein (KCN) are part of the casein micelle complex in milk. Both BCN and KCN are amphiphillic proteins with a molar mass of 24 and 19 kDa and carry net charges of -14 and -4, respectively. Both BCN and KCN form soap-like micelles, with 40 and 65 monomers, respectively. The net negative charges are located in the corona of the micelles. On mixing LF with the caseins, coacervates are formed. We analyzed the structure of these coarcervates using SAXS. It was found that LF binds to the corona of the micellar structures, at the charge neutrality point. BCN/LF and KCN/LF ratios at the charge neutrality point were found to be ~1.2 and ~5, respectively. We think that the findings are relevant for the protection mechanism of globular proteins in bodily fluids where unstructured proteins are abundant (saliva). The complexes will prevent docking of enzymes on specific charged groups on the globular protein.

Β-lactoglobulin self-assembly: structural changes in early stages and disulfide bonding in fibrils.

Bovine ß-lactoglobulin (ß-Lg) self-assembles into long amyloid-like fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). Heating ß-Lg under fibril-forming conditions shows a lag phase before fibrils start forming. We have investigated the structural characteristics of ß-Lg during the lag phase and the composition of ß-Lg fibrils after their separation using ultracentrifugation. During the lag phase, the circular dichroism spectra of heated ß-Lg showed rapid unfolding, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of samples showed increasing hydrolysis of ß-Lg. The SDS-PAGE profiles of fibrils separated by ultra centrifugation showed that after six hours, the fibrils consisted of a few preferentially accumulated peptides. Two-dimensional SDS-PAGE under reducing and nonreducing conditions showed the presence of disulfide-bonded fragments in the fibrils. The sequences in these peptide bands were characterized by in-gel digestion electrospray ionization (ESI)-MS/MS. The composition of solubilized fibrils was also characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS/MS. Both MS analyses showed that peptides in fibrils were primarily from the N-terminal region, although there was some evidence of peptides from the C-terminal part of the molecule present in the higher molecular weight gel bands. We suggest that although the N-terminal region of ß-Lg is almost certainly involved in the formation of the fibrils, other peptide fragments linked through disulfide bonds may also be present in the fibrils during self-assembly.

Coacervates of lysozyme and ß-casein.

Complexes are formed when positively charged lysozyme (LYZ) is mixed with negatively charged caseins. Adding ß-casein (BCN) to LYZ leads to flocculation even at low addition levels. Titrating LYZ into BCN shows that complexes are formed up to a critical composition (x=[LYZ]/([LYZ]+[BCN]). The formation of these complex coacervates increases asymptotically toward the molar charge equivalent ratio (xcrit), where the size of the complexes also seems to grow asymptotically. At xcrit, insoluble precipitates of charge-neutral complexes are formed. The precipitates can be re-dispersed by adding NaCl. The value of xcrit shifts to higher values on the LYZ side with increasing salt concentration and pH. Increasing the pH, de-protonates the BCN and protonates the LYZ, and therefore, charge neutrality will shift toward the LYZ side. xcrit increases linearly from 0.2 at no salt to 0.5 at 0.5M NaCl. It ends abruptly at a salt concentration of 0.5M after which a clear mixed solution remains. Away from the charge equivalent ratio, it seems that the buildup of charges limits the complex size. A simple scaling law to predict the size of the complex is proposed. By assuming that surface charge density is constant or can reach only a maximum value, it follows that scattering intensity is proportional to |(1-x/xcrit)|(-3) where x is the mole fraction of one protein and xcrit the value of the mole fraction at the charge equivalent ratio. Both scattering intensity and particle size seem to obey this simple assumption. For BCN-LYZ, the buildup occurs only at the LYZside in contrast to lactoferrin which forms stable complexes on either side of xcrit. The reason that the complexes are formed at the BCN side only may be due to the small size of LYZ, which induces a bending energy in the BCN on adsorption.

The protein interactions and rheological properties of skim milk heated in the presence of low levels of reducing agent.

Skim milk with low levels of added ß-mercaptoethanol (SM-ME) and untreated skim milk (SM) were heated and then made into acid gels. Acid gels prepared from heated SM-ME had markedly higher firmness and contained more protein connections than acid gels prepared from heated SM. Electrophoretic analyses of the milks showed that the levels of ß-lactoglobulin and α-lactalbumin associated with the casein micelles increased with increasing ß-ME concentration. The levels of disulphide-linked whey proteins were higher in SM-ME than in SM. This suggested that there may be higher levels of initiators for thiol-disulphide exchange reactions, resulting in an increase in the rate of the reactions and the formation of greater numbers of small aggregates, in SM-ME than in SM. Consequently, acid gels made from SM-ME may have more bonds and more particles participating in the network, resulting in firmer gels, than acid gels made from SM.

Pressure-induced denaturation of ß-lactoglobulin in skim milk: effect of milk concentration.

The effect of milk concentration (10-40% TS) on the kinetics of the pressure-induced denaturation of ß-lactoglobulin (ß-LG) was studied. The denaturation was found to be a second-order process at all milk concentrations and pressures. There was a change in pressure dependence of the rate constants for denaturation at about 300 MPa, and this effect became more pronounced as the milk concentration increased. At pressures ≥300 MPa, a small effect of milk concentration was observed, with small decreases in the rate of denaturation as the milk concentration was increased above 20% TS. This was attributed to the lower pH as the milk concentration was increased. In contrast, at 200 MPa, ß-LG denaturation was markedly retarded as the milk solids concentration was increased. This was attributed to the increased lactose concentration at higher milk concentrations. This would promote ß-LG dimerization at this pressure and this would stabilize the ß-LG to denaturation.

Whey protein nanofibrils: the environment-morphology-functionality relationship in lyophilization, rehydration, and seeding.

Amyloid-like fibrils from ß-lactoglobulin have potential as efficient thickening and gelling agents for food and biomedical applications, but the link between fibril morphology and bulk viscosity is poorly understood. We examined how lyophilization and rehydration affects the morphology and rheological properties of semiflexible (i.e., straight) and highly flexible (i.e., curly) fibrils, the latter made with 80 mM CaCl(2). Straight fibrils were fractured into short rods by lyophilization and rehydration, whereas curly fibrils sustained little damage. This was reflected in the viscosities of rehydrated fibril dispersions, which were much lower for straight fibrils than for curly fibrils. Lyophilized straight or curly fibrils seeded new fibril growth, but viscosity enhancement due to seeding was negligible. We believe that the increase in fibril concentration caused by seeding was counterbalanced by a decrease in fibril length, reducing the ability of fibrils to form physical entanglement networks.

Effects of adding low levels of a disulfide reducing agent on the disulfide interactions of ß-lactoglobulin and κ-casein in skim milk.

Low concentrations of a disulfide reducing agent were added to unheated and heated (80 °C for 30 min) skim milk, with and without added whey protein. The reduction of the ß-lactoglobulin and κ-casein disulfide bonds was monitored over time using electrophoresis. The distribution of the proteins between the colloidal and serum phases was also investigated. κ-Casein disulfide bonds were reduced in preference to those of ß-lactoglobulin in both unheated and heated skim milk (with or without added whey protein). In addition, in heated skim milk, while the serum κ-casein was reduced more readily than the colloidal κ-casein, the distribution of κ-casein between the two phases was not affected.

Interaction of lactoferrin and lysozyme with casein micelles.

On addition of lactoferrin (LF) to skim milk, the turbidity decreases. The basic protein binds to the caseins in the casein micelles, which is then followed by a (partial) disintegration of the casein micelles. The amount of LF initially binding to casein micelles follows a Langmuir adsorption isotherm. The kinetics of the binding of LF could be described by first-order kinetics and similarly the disintegration kinetics. The disintegration was, however, about 10 times slower than the initial adsorption, which allowed investigating both phenomena. Kinetic data were also obtained from turbidity measurements, and all data could be described with one equation. The disintegration of the casein micelles was further characterized by an activation energy of 52 kJ/mol. The initial increase in hydrodynamic size of the casein micelles could be accounted for by assuming that it would go as the cube root of the mass using the adsorption and disintegration kinetics as determined from gel electrophoresis. The results show that LF binds to casein micelles and that subsequently the casein micelles partly disintegrate. All micelles behave in a similar manner as average particle size decreases. Lysozyme also bound to the casein micelles, and this binding followed a Langmuir adsorption isotherm. However, lysozyme did not cause the disintegration of the casein micelles.

Effect of calcium on the morphology and functionality of whey protein nanofibrils.

Self-assembly of amyloid-like nanofibrils during heating of bovine whey proteins at 80 °C and pH 2 is accelerated by the presence of NaCl and/or CaCl(2), but the rheological consequences of accelerated self-assembly are largely unknown. This investigation focused on the impact of CaCl(2) on the evolution of rheological properties and fibril morphology of heated whey protein isolate (WPI), both during self-assembly at high temperature and after cooling. Continuous rotational rheometry of heated 2% w/w WPI showed a nonlinear effect of CaCl(2) on the viscosity of fibril dispersions, which we attributed to effects on fibril flexibility and thus the balance between intrafibril and interfibril entanglements. Small-amplitude oscillatory measurements made in situ during heating of 10% w/w WPI at 80 °C suggest that CaCl(2) is not involved in either fibril structure or gel structure, and this was confirmed with dialysis experiments.